The proposed Mentored Clinical Scientist Development Award aims to support the development of a talented candidate into an independent physician-scientist, while advancing promising preliminary work to improve the diagnosis and management of congenital adrenal hyperplasia (CAH). CAH comprises a set of autosomal recessive genetic defects in cortisol biosynthesis, and 21-hydroxylase deficiency (21OHD) accounts for >95% of CAH cases. With a prevalence of 1:1000 in its nonclassic form, a defined monogenic origin, and circumscribed biochemical basis, 21OHD represents a paradigm for genetic disorders of metabolism. Scientific progress on steroid flux and physiology in 21OHD, however, has been stagnant for decades. Unreliable steroid intermediates and final products in major pathways identified in the 1950s are still used to diagnose and to monitor disease, which hampers efforts to provide optimal medical care and to develop better treatments. These currently used biomarkers correlate poorly with clinical evidence of adrenal androgen excess and also derive from the gonad, further limiting their utility in adults with 21OHD. The long-term goals of the proposed research are 1) to develop improved methods to diagnose 21OHD, including nonclassic disease and 2) to define the steroids responsible for clinical manifestations of androgen excess in these patients, which will enhance treatment monitoring. For Aim 1, we hypothesize that a panel of steroid biomarkers upstream the enzymatic defect will accurately diagnose classic and nonclassic 21OHD in a single random blood draw. We will employ liquid chromatography-tandem mass spectrometry (LC-MS/MS) to comprehensively characterize steroids in patients with classic and nonclassic 21OHD compared to unaffected individuals. For Aim 2, we hypothesize that adrenal-specific 11-oxygenated androgens are primarily responsible for the androgen excess of 21OHD. With the help of LC-MS/MS, we will generate a detailed characterization of the adrenal androgen precursors flux in patients with 21OHD, and by using an in vitro androgen receptor model linked to a luciferase reporter, we will define the active androgens in 21OHD. All studies will be conducted at the University of Michigan, which provides a rich and rigorous research environment, ideal mentorship and abundant resources for the completion of the proposed studies. Future directions include validation of the biomarkers emerging from these studies in prospective multicenter trials, by assessing their response to treatment. The candidate will pursue additional training in genetics, study design, and advanced steroid metabolomics, which will fully prepare her to become a lead scientist in CAH and other disorders of steroid metabolism.